Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells

Lauri J. Reuter, Michael J. Bailey, Jussi J. Joensuu, Anneli Ritala

Research output: Contribution to journalArticleScientificpeer-review

31 Citations (Scopus)

Abstract

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.

Original languageEnglish
Pages (from-to)402-410
Number of pages9
JournalPlant Biotechnology Journal
Volume12
Issue number4
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

Recombinant Proteins
recombinant proteins
cell suspension culture
Tobacco
Suspensions
tobacco
Plant Cells
water
protein bodies
Cell Culture Techniques
commercialization
cells
bioreactors
green fluorescent protein
production costs
surfactants
Technology
purity
Costs and Cost Analysis
cell culture

Keywords

  • Aqueous two-phase separation
  • Bioreactor
  • Hydrophobin fusion
  • Scale-up, suspension cells
  • Tobacco bright yellow 2

Cite this

@article{2dd9599c2ed9401e82e09aaa32f93980,
title = "Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells",
abstract = "Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60{\%} recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.",
keywords = "Aqueous two-phase separation, Bioreactor, Hydrophobin fusion, Scale-up, suspension cells, Tobacco bright yellow 2",
author = "Reuter, {Lauri J.} and Bailey, {Michael J.} and Joensuu, {Jussi J.} and Anneli Ritala",
year = "2014",
month = "1",
day = "1",
doi = "10.1111/pbi.12147",
language = "English",
volume = "12",
pages = "402--410",
journal = "Plant Biotechnology Journal",
issn = "1467-7644",
publisher = "Wiley",
number = "4",

}

Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. / Reuter, Lauri J.; Bailey, Michael J.; Joensuu, Jussi J.; Ritala, Anneli.

In: Plant Biotechnology Journal, Vol. 12, No. 4, 01.01.2014, p. 402-410.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells

AU - Reuter, Lauri J.

AU - Bailey, Michael J.

AU - Joensuu, Jussi J.

AU - Ritala, Anneli

PY - 2014/1/1

Y1 - 2014/1/1

N2 - Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.

AB - Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.

KW - Aqueous two-phase separation

KW - Bioreactor

KW - Hydrophobin fusion

KW - Scale-up, suspension cells

KW - Tobacco bright yellow 2

UR - http://www.scopus.com/inward/record.url?scp=84899059237&partnerID=8YFLogxK

U2 - 10.1111/pbi.12147

DO - 10.1111/pbi.12147

M3 - Article

C2 - 24341724

AN - SCOPUS:84899059237

VL - 12

SP - 402

EP - 410

JO - Plant Biotechnology Journal

JF - Plant Biotechnology Journal

SN - 1467-7644

IS - 4

ER -